Significance: Developed quantum mechanics/molecular mechanics (QM/MM) supercomputation is used to identify maturation of immunoglobulin (Ig) reactivity through optimization of amino acids in the catalytic core, focused on enhanced nucleophilic attack on phosphorus. A computationally optimized variant, l -Leu47Lys, delivers 340-fold reactivity enhancement for paraoxon relative to wild-type Ig-paraoxonase resulting from substrate-induced ionization of the Tyr37 nucleophile. Variant antibody stereoselectivity toward a P-chiral phenylphosphonate toxin results from starkly contrasting solvent-assisted or general base catalytic mechanisms relative to the parent Ig-paraoxonase. These mechanisms are defined by crystallographic and QM/MM analyses as resulting from changed substrate orientation and participating amino acid conformations within the catalytic site.

The substrate-driven closure determines an outstanding efficacy of antibiotic inactivation in a new kinase subfamily.

Peptides are promising drug candidates due to high specificity and standout safety. Identification of bioactive peptides de novo using molecular docking is a widely used approach. However, current scoring functions are poorly optimized for peptide ligands. In this work, we present a novel algorithm PeptoGrid that rescores poses predicted by AutoDock Vina according to frequency information of ligand atoms with particular properties appearing at different positions in the target protein’s ligand binding site. We explored the relevance of PeptoGrid ranking with a virtual screening of peptide libraries using angiotensin-converting enzyme and GABAB receptor as targets. A reasonable agreement between the computational and experimental data suggests that PeptoGrid is suitable for discovering functional leads.

Butyrylcholinesterase (BChE) is considered as an efficient stoichiometric antidote against organophosphorus (OP) poisons. Recently we utilized combination of calculations and ultrahigh-throughput screening (uHTS) to select BChE variants capable of catalytic destruction of OP pesticide paraoxon. The purpose of this study was to elucidate the molecular mechanism underlying enzymatic hydrolysis of paraoxon by BChE variants using hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. Detailed analysis of accomplished QM/MM runs revealed that histidine residues introduced into the acyl-binding loop are always located in close proximity with aspartate residue at position 70. Histidine residue acts as general base thus leading to attacking water molecule activation and subsequent SN2 inline hydrolysis resulting in BChE reactivation. This combination resembles canonical catalytic triad found in active centers of various proteases. Carboxyl group activates histidine residue by altering its pK_a, which in turn promotes the activation of water molecule in terms of its nucleophilicity. Observed re-protonation of catalytic serine residue at position 198 from histidine residue at position 438 recovers initial configuration of the enzyme’s active center, facilitating next catalytic cycle. We therefore suggest that utilization of uHTS platform in combination with deciphering of molecular mechanisms by QM/MM calculations may significantly improve our knowledge of enzyme function, propose new strategies for enzyme design and open new horizons in generation of catalytic bioscavengers against OP poisons.

The cysteine protease from the tobacco etch virus (TEVp) is a well-known and widely utilized enzyme. TEVp's chymotrypsin-like fold is generally associated with serine catalytic triads that differ in terms of a reaction mechanism from the most well-studied papain-like cysteine proteases. The question of what dominates the TEVp mechanism, nucleophile identity, or structural composition has never been previously addressed. Here, we use enhanced sampling multiscale modeling to uncover that TEVp combines the features of two worlds in such a way that potentially hampers its activity. We show that TEVp cysteine is strictly in the anionic form in a free enzyme similar to papain. Peptide binding shifts the equilibrium toward the nucleophile ' s protonated form, characteristic of chymotrypsin-like proteases, although the cysteinyl anion form is still present and interconversion is rapid. This way cysteine protonation generates enzyme states that are a diversion from the most effective course of action, with only 13.2% of Michaelis complex sub-states able to initiate the reaction. As a result, we propose an updated view on the reaction mechanism catalyzed by TEVp. We also demonstrate that AlphaFold is able to construct protease-substrate complexes with high accuracy. We propose that our findings open a way for its industrious use in enzymological tasks. Unique features of TEVp discovered in this work open a discussion on the evolutionary history and trade-offs of optimizing serine triad-associated folds to cysteine as a nucleophile.